Our ultimate goal is to determine the physiological function and molecular mechanism of myosin-l. Myosin-l is thought to play an integral role in the structure and dynamics of the plasma membrane and intracellular membranes. However, determination of the precise physiological functions of myosin-l have been complicated by the fact that a single cell expresses several similar isoforms. It is not known if each isoform has unique biochemical and structural properties suitable for a specific function, or if the isoforms are functionally redundant. Therefore, our efforts in this proposal are focused on characterizing the chemical, structural, and dynamic properties of the myosin-l isoforms within a single cell type, and correlating possible isoform-specific properties with physiological function. We will obtain a physical framework in which to discuss myosin-l function by investigating the enzymatic, force-producing, and assembly properties of each isoform, and we will investigate the in vivo dynamics and organization of the myosin-l isoforms in live cells using high- resolution fluorescence microscopy. Acanthamoeba castellanii myosin-l isoforms were the first myosin-ls discovered, and their cellular localization and enzymatic activities are the most extensively characterized. Recent advances in Acanthamoeba transfection makes it an ideal organism for cell biological studies. Therefore, to make rapid progress in the understanding of membrane-based motility in all eukaryotic cells, we are addressing the following specific aims using Acanthamoeba myosin-l: (1) Determine the enzymatic and force producing properties of myosin-l isoforms. (2) Determine the structural and associative properties of myosin-l isoforms. (3) Measure the cellular dynamics of each myosin-l isoform.